The overall goal of the proposal is to elucidate the mechanism of viral uncoating for nonenveloped DNA viruses using Simian Virus 40 (SV40) as a model virus. Viruses replicate within living cells by exploiting the host cellular machinery. They are internalized by binding to their specific host cell surface receptor and pirating established endocytic pathways. Upon internalization, the virus must shed its membrane encapsulation acquired during endocytic entry. To accomplish this, viruses have evolved mechanisms to penetrate or permeabilize the endocytic membrane, or to transport their genome across membranes. Some viruses, including SV40 are transported to the endoplasmic reticulum (ER). During this process, the virus loses its endocytic membrane coat and is released into the ER lumen where it acquires the larger organelle membrane barrier. Recent data suggests that within the ER the virus disassembles and its genome is delivered to the cytosol/nucleus. This facilitates genome replication, the cytosolic synthesis of viral proteins, and viral assembly. Both the mechanisms of viral uncoating and genome penetration are largely unknown. The ER is a specialized maturation compartment that supports the folding and assembly of thousands of membrane embedded and soluble secretory proteins. The ER also possesses a quality control activity that ensures that only properly folded and assembled proteins are exported through the secretory pathway. Resident ER proteins dedicated to the maturation and quality control of secretory cargo are likely usurped by several nonenveloped viruses for the uncoating of their icosohedral capsids. The ER is a tightly controlled organelle that is able to remodel itself to alter the balance between maturation and quality control through the unfolded protein response signal transduction pathway. We have recently determined that the SV40 minor structural proteins VP2 and VP3 are capable of post- translationally inserting into ER membranes. These proteins appear to possess viroporin activity since they permeabilize bacterial membranes supporting a potential role for VP2 and VP3 in viral genome penetration of the ER membrane. We hypothesize that during entry the virus disassociates in the ER with the help of resident chaperones and quality control factors. Its genome along with viral proteins are then retrotranslocated from the ER to the cytosol/nucleus in a process that involves ER resident proteins and the membrane inserted VP2 and VP3, which are liberated following particle disassembly or uncoating. The specific aims of this proposal are: (1) to explore the affect of SV40 infection on ER physiology or function;(2) to determine the status of the viral components (genome, major and minor structural proteins) after entry into the ER;and (3) to investigate the mechanism of genome penetration of the ER membrane using a newly developed in vitro retrotranslocation assay. Together, these studies will provide valuable insight into the uncoating and penetration processes for nonenveloped viruses. PUBLIC HEALTH RELEVANCE: These studies will provide valuable insight into two essential steps in the life cycle of nonenveloped viruses;uncoating and penetration. The employment of Simian Virus 40 (SV40) as a model virus to characterize the mechanisms utilized by the virus to disassemble within the cell and translocate its genome to its site of replication will provide us with a deeper understanding of the biology for this small DNA tumor virus and its related human viruses BK, JC, the newly discovered KI and WU viruses, as well as papilloma viruses, which are associated with cervical cancer. Furthermore, understanding the viral-mediated DNA delivery process also has potential applications for increasing the efficiency of DNA delivery vehicles for gene therapy.